Microparticles

ABSTRACT

The invention provides a blood substitute product comprising haemoglobin and a self-assembled microparticle having an acid having two or more acid groups and an organic base in a solvent. The particle is of micron scale. The microparticle may be obtained by contacting a bis-acid and organic base in a hydrophilic solvent, wherein the acid is insoluble or sparingly soluble in the hydrophilic solvent and the organic base is soluble in a hydrophilic solvent.

This invention relates to a microparticles, in particular toself-assembled microparticles and their use as a component of anartificial blood product or a blood substitute and a method of preparingthe microparticles. The microparticles and porous materials are usefulin a wide range of physical and chemical processes especially wherecirculation in the blood stream is required.

Blood surrogates, also referred to as artificial blood, bloodsubstitutes or oxygen carrying substitutes are of immense importance incircumstances where immediate blood supply is required but cannot besupplied through traditional blood transfusion. This can be for examplein cases of haemophilia, in trauma units, in cases where blood can becontaminated by disease, during transplant surgery, or in remotelocations away from medical facilities, for example on the battlefieldand in road traffic accidents. A major purpose behind blood substitutesis the elimination of immune response, often seen in donor blood, andelimination of disease transmission. Religious objection to bloodtransfusion is also a limiting factor with donated blood. Bloodsubstitutes can also be used for the storage and preservation of donororgans and other body tissues.

It is also essential that the manufacturing process and cost of theultimate substitute is cost effective. A 60 g unit of donor bloodtypically costs a hospital $300 therefore the price of a bloodsubstitute would need to be less than $5 per gram or provide acompelling reason for use.

Investigation into development of viable blood substitutes has beenongoing for more than 70 years. The primary driver, the transport ofoxygen by red blood cells has steered most research into the developmentof haemoglobin-based oxygen carriers (HBOCs). The chemical carrier ofoxygen in haemoglobin is protoporphyrin IX which has more recentlystimulated additional research into porphyrin based technologies.

Microparticles as blood substitutes are known, for example as describedin WO9629346, WO2006108047 U.S. Pat. No. 6,498,141, HK1207560, U.S. Pat.Nos. 5,770,727 5,387,672.

Known blood substitutes suffer from a number of disadvantages. Some sideeffects reported include transient yellow skin discoloration, nausea,mild to moderate increase in blood pressure (10 to 20 mm/Hg), vomiting,low urine output, difficulty swallowing, flatulence, and low red bloodcell count. Existing methodology for preparation of HBOCs suffers fromcomplex manufacturing processes and result in polymers with broad rangesin size or heterogeneous polymeric forms of haemoglobin. The processesfor manufacture often include complex organic chemistry, laboriousdialysis and/or chromatographic separation technologies which aredifficult to scale up. The current HBOCs in trials or development areoften cross-linked with cytotoxic chemicals including, for examplealdehydes such as glutaraldehyde where the mechanism of crosslinking iswell known to be random, ill-defined and difficult to reproduce. It ispostulated that dimeric forms of haemoglobin may be the cause of toxiceffects in some HBOCs, however, the methods of cross-linking describedin other HBOCs could be responsible for toxicity.

It is known that haemoglobin can be cross-linked with bis-carboxy fattyacids. However known cross-linked haemoglobins disadvantageously have abroad molecular weight range and require complex and laboriouspurification processes including gel permeation, anion exchange andcation exchange chromatography to provide a usable product.

We have now found that these and other problems associated with knownblood substitute products may be ameliorated by providing a particulatesupport comprising a self-assembled microparticle comprising a fattyacid having two or more carboxylic acid groups and a base which providea narrow particle size distribution or macroporous material formed bycontacting self-assembled microparticles. The microparticles aresuitably biodegradable.

In a first aspect, the invention provides a blood substitute compositioncomprising mammalian haemoglobin and a self-assembled microparticle.Suitably the microparticle comprises an acid having two or more acidgroups and an organic base which is soluble in a hydrophilic solvent.

Suitably, the blood substitute composition comprises a stablepolymerized haemoglobin solution, comprising mammalian hemoglobincross-linked with a self-assembled microparticle.

The term “blood-substitute” as employed herein denotes a HBOCcomposition for use in humans, mammals and other vertebrates. The HBOCis capable of transporting and transferring oxygen to vital organs andtissues. A vertebrate is defined to include humans, or any othervertebrate animals which uses blood in a circulatory system to transferoxygen to tissue. By way of examples a preferred vertebrate fortreatment with the substance of this invention is a mammal, such as aprimate, a canine, a feline, an equine, a porcine, a bovine, an ovine, arodent or an avian. A vertebrate treated with the substance of thisinvention can be fetal, post-natal vertebrate, or a vertebrate at timeof birth. The HBOC substance described herein may also be used for thestorage and preservation of donor organs and mammalian tissues.

The use of a self-assembled microparticle enables a polymer ofhaemoglobin, of a regular size and shape and narrow molecular weightdistribution to be produced. The microparticle contains naturalmaterials, which are cross-linked by amide bonds and are thereforebiodegradable by protease activity. As such, they are likely to be oflittle, or no concern to physiological functions. The presentmicroparticles are advantageously monodispersed and small enough totravel through the smallest of capillaries where oxygen transfer occurs.

The weight to weight ratio of the haemoglobin to cross-linking agent aresignificantly lower than hitherto known. Known products have a molecularweight in the range of 1.7×10⁷.

The present invention provides microparticles having a molecular weightof at least 1.0×10⁸, preferably from 1.6×10¹¹ to 2×10¹³ for example4×10¹².

Haemoglobin from any suitable source may be used to prepare the bloodsubstitute of the present invention. Examples include old or outdatedhuman blood, bovine blood, ovine blood, porcine blood, equine blood, andblood from other vertebrates. Transgenic haemoglobin, such as thetransgenic haemoglobin described in EIO/TECHNOLOGY, 1z: 55-59 (1994) andrecombinant haemoglobin (Nature, 356:258-60 (1992)) can also be used.

The microparticle acid suitably comprises a bis-acid, preferably abis-aliphatic acid and suitably comprises two or more carboxylic acidgroups, although other acid groups may be employed. Suitably thebis-acid is insoluble or sparingly soluble in the hydrophilic solvent.Suitably, by contacting the acid, preferably bis-aliphatic acid with anorganic base which is soluble in the hydrophilic solvent, the acid maybe solubilised.

The solvent is suitably hydrophilic, preferably an aqueous solution, forexample a water in oil emulsion within an aqueous phase, and especiallywater. Advantageously, an aqueous-based solvent, preferably water,allows the microparticle to be used in applications in whichenvironmental considerations are important. For example, themicroparticle may be formulated into an aqueous-based product which maybe suitable for personal use or consumption, medical uses and forexample as a biocide.

In the preferred embodiment the bis-aliphatic acid comprises abis-carboxylic fatty acid in which terminal carboxylic acids are linkedby a region which is less hydrophilic than the terminal carboxylic acidsand is preferably hydrophobic. The less hydrophilic region may comprisea backbone with substituents and/or the backbone may compriseheteroatoms, for example poly-epsilon lysine. Preferably the regionlinking the carboxylic acids is hydrophobic and preferably a hydrocarbylgroup. In an especially preferred embodiment, the hydrophobic group isan aliphatic hydrocarbyl group. Preferably, the bis-acid comprises acompound of general formula HOOC—(CH₂)_(n)—COOH wherein n issufficiently large that the bis acid is sparingly soluble or insolublein water. Preferably n is at least 5, more preferably at least 6,especially at least 7. Suitably n is not more than 40, preferably notmore than 36, more preferably not more than 25, and especially not morethan 20. Preferably n is from 7 to 18.

In a preferred embodiment, the organic acid comprises a C₇ to C₁₈ biscarboxylic fatty acid. In another preferred embodiment, the organic acidcomprises a C₇ to C₁₃ bis carboxylic fatty acid together with a furtheracid selected from a EDTA, nitrolotriacetic acid and a monocarboxylicacid, preferably a C₆ to C₁₈ carboxylic acid, for example caproic acid,palmitic acid and octanoic acid.

By selecting more than one acid for example in which the acids havedifferent n values, the size of the microparticle may be tailored. Alonger hydrophobic portion connecting the acid groups suitably providesa larger microparticle. For example where n is 8, sebacic acid, aparticle of size 2.6 microns may be obtained and where n is 11,brassylic acid, a particle of size 3.0 microns may be obtained.

The bis-carboxy fatty acid can also be unsaturated for example traumaticacid, or substituted or both unsaturated and substituted. Suitably, thesubstitution does not cause the bis-acid to be soluble in aqueoussolution. When the bis-aliphatic acid is contacted with the aid of asolvent soluble organic base, microparticles are formed spontaneously.

The bis-aliphatic acid may comprise: a bis-phosphonic acid of generalformula (HO)₂OP—(CH₂)_(n)—PO(OH)₂ or an unsaturated bis-phosphonic acid;a mono-carboxylic mono-phosphonic acid of general formulaHOOC—(CH₂)_(n)—PO(OH)₂ or an unsaturated version of such bis-acid; abis-sulfonic acid of general formula (HO)O₂S—(CH₂)_(n)—SO₂(OH) or anunsaturated version of such bis-acid; a mono-carboxylic mono-sulfonicacid of general formula HOOC—(CH₂)_(n)—SO₂(OH) or an unsaturated versionof such a bis-acid; a bis-boronic acid of general formula(HO)₂B—(CH₂)_(n)—B(OH)₂ or an unsaturated bis-boronic acid, orsubstituted bis-boronic acid; a mono-carboxylic mono-boronic acid ofgeneral formula HOOC—(CH₂)_(n)—B(OH)₂ an unsaturated version of suchbis-acid; or a substituted version of said bis-acids. In these acids, nis sufficiently large that the bis acid is sparingly soluble orinsoluble in water. Preferably n is at least 5, more preferably at least6 and especially at least 7. Suitably n is not more than 40, preferablynot more than 36 more preferably not more than 25, and especially notmore than 20. Preferably n is from 7 to 18.

Suitably, the organic base combines with the bis-acid moieties such thatthe combination of the two components comprises two separate hydrophilicor ionic head regions connected by a hydrophobic region. Without wishingto be bound by theory, it is believed that the hydrophobic regions andhydrophilic regions of adjacent bis-acids with organic base align toform micelles and lead to self-assembly of the microparticles of theinvention. Preferably, the microparticle comprises a multi-lamellarstructure in which further molecules comprising the bis-acids with theorganic base, align with the hydrophilic head of anotherbis-acid/organic base so as to form a multi-lamellar structure.

The organic base may be selected from a range of bases which, togetherwith the bis-acid forms a self-assembling microparticle. Preferably, theorganic base comprises an amine, suitably an aliphatic amine or anaromatic amine having a basic character or other nitrogen-containingbase. Examples of suitable organic bases include alkylated amines andpolyamines including amines having one or two C₁₋₄ N-alkyl -groups, forexample methylated amines. Examples of preferred amines includeN-methylmorpholine, 4-methylmorpholine (NMM), N,N-dimethylaminoethanol(DMAE), 4-dimethylaminopyridine (DMAP), imidazole or 1-methylamidazole,poly(diallyldimethylammonium chloride) (PDAC), didecyldimethylammoniumchloride (DDAC) and dodecyldipropylenetriamine (DDPT).

In preferred embodiments, the acid is suitably one or more of brassylicacid, sebacic acid and azelaic acid in combination with a base selectedfrom methylmorpholine (NMM), N,N-dimethylaminoethanol (DMAE),4-dimethylaminopyridine (DMAP), imidazole, 1-methylamidazole,poly(diallyldimethylammonium chloride) (PDAC), didecyldimethylammoniumchloride (DDAC) and dodecyldipropylenetriamine (DDPT).

Preferred examples include microparticles comprising brassylic acid andPDAC, brassylic acid and DDAC, brassylic acid and DDPT, sebacic acid andNMM, poly epsilon lysine in combination with one or more of sebacicacid, brassylic acid and azelaic acid.

We have found that microparticles according to the invention comprisingamines having antimicrobial properties are particularly suited for useas antimicrobial compositions and biocides. The level of antimicrobialactivity of the base may be higher when in the form of a self-assembledmicroparticle according to the invention as compared to when in aconventional formulation.

According to a further aspect the invention also provides the use of aself-assembled microparticle comprising a bis acid and a water-solublebase, the base being displaceable by a protein and for covalentcross-linking.

In a further aspect, the invention provides a self-assembledmicroparticle comprising an acid having two or more acid groups,preferably a bis acid, covalently bonded to a haemoglobin molecule.

The covalently bonded haemoglobin is suitably produced by addinghaemoglobin to a self-assembled microparticle comprising an acid havingtwo or more acid groups and a water-soluble base which is displaceablein part or whole by haemoglobin.

The protein suitably comprises haemoglobin either individually or incombination with one or more other proteins, for example a catalase anda superoxide dismutase.

The acid and base are suitably combined in relative quantities such thatthe molar ratio of acid groups in the acid to basic groups in the baseis approximately stoichiometriuc such that self-assembled microparticlesform. The molar quantity of acid groups to base groups may be less ormore than stoichiometric provided the self-assembled particles form.Where the ratio of acid groups to base groups is too low or too high,-assembled particles do not form as the excess component disruptsstructure of the acid and base. The ratio of acid groups to basic groupsthat allow formation of the self-assembled particle will vary dependingon the particular acid and particular base.

The skilled person will be able to determine whether a self-assembledparticle is formed by observing under a microscope with magnification ata level to visually observe particles for example at 40× magnification.The relative quantities of the acid and base will be able to be modifiedto determine the minimum and maximum ratio of the components at whichmicroparticles form. Acids having longer chains may providemicroparticles which are more stable than microparticles (with the samebase and same molar ratio) comprising an acid having a shorter chain.The greater stability may allow a lower level of acid to be employed anda lower ratio of acid groups to basic groups may still allow amicroparticle to form.

Suitably, the ratio of acid groups to basic groups in the acid and baseis from 0.6 to 1.4:1, preferably 0.7 to 1.3:1, more preferably 0.8 to1.2:1 and desirably 0.9 to 1.1:1. Sebacic acid and brassylic acid areexamples of preferred acids. Suitably a microparticle comprising sebacicacid with a base has a ratio of sebacic acid to base of 0.85 to 1.15:1.A microparticle comprising brassylic acid with a base has a ratio ofbrassylic acid to base of 0.8 to 1.2:1. In a preferred embodiment, theacid and base are present at levels to provide a molar ratio of acidgroups to basic groups of 1:1.

The organic base may be reactive so as to enable cross-linking of theself-assembled microparticles for form a macroporous material. Theorganic base need not be reactive in which case it may suitably bedisplaced by another reactive species to allow subsequent cross-linkingto form a macroporous material. The solvent soluble organic base can bedisplaced by addition of a reactive species including, but not limitedto, amine containing organic components. The amine suitably allowscross-linking of the microparticles by amide bond formation. In thepreferred embodiment the amine containing organic component is apolymeric amine including but not limited to a peptide, protein,polyallylamine, polyethyleneimine and other polyamines.

Examples of suitable amines and polyamines include ethylenediamine,poly-e-lysine, polyallylamine, polyethyleneimine,aminopropyltrialkoxysilanes,3-(2-aminoethylamino)propyltrimethoxysilane,N-(3-(trimethoxysilyl)-propyl)diethyenetriamine.

In formation of the microparticle or macroporous material the aboveaforementioned bis acids may be mixed in any proportions. In addition,the reactive amines may also be mixed.

Suitably, the microparticles comprise functional components, tailoredaccording to the intended use.

A self-assembled microparticle or macroporous material according to theinvention may also comprise a functional material supported by thepolymer. Examples of suitable functional materials include a catalyst,an initiator species for peptide synthesis or oligonucleotide synthesis,a pharmaceutical active, an agrochemical active, a macromolecule, anenzyme, a nucleic acid sequence and a protein.

The invention is particularly useful in supporting precious metalcatalysts, for example palladium catalysts. A particular advantageousexample is palladium.

The self-assembled microparticle may be produced by a method comprisingcontacting the acid having two or more acid groups with an organic basein an aqueous medium, preferably water.

Suitably the polymerisation and cross-linking is initiated by processesknown to those skilled in the art. For example, self-assembledmicroparticles prepared in water with an amine containing component canbe cross-linked using a water soluble carbodiimide.

Suitably, the self-assembled microparticle material according to theinvention is substantially mono-disperse. That is the material hasparticles which are all substantially the same size. Monodispersemicroparticles may advantageously travel in the blood stream withouttransferring across capillary walls or blocking capillaries, effectivelybehaving in a similar manner to an erythrocyte.

The self-assembled microparticles of the present invention may be usedin separate or combined processes, for example, drug delivery incombination with oxygen transport.

The self-assembled microparticles may be used as a carrier to carry acompound which is to be released over a period of time, for example apharmaceutical. This use provides a means of tailoring a dosage regimeof the compound according to the loading of the compound in the support.In the case of a pharmaceutical, this may be advantageous in assistingthe correct dosage of an active, for example with continuous slowrelease rather than requiring a patient to take periodic large doses.

The microparticles of the invention may be formulated into a compositionfor a wide-range of variations in use, including a compositioncontaining haemoglobin in combination with other proteins or enzymessuch as a catalase, or in combination with compounds that provide andallosteric effect such as 2,3-bisphosphoglyceric acid.

The invention is illustrated by the following non-limiting examples.

EXAMPLE 1—PREPARATION OF SELF-ASSEMBLED MICROPARTICLES

Brassylic acid (1.54 g, 6.31 mmol) and 4-dimethylaminopyridine (DMAP,1.54 g, 12.62 mmol) were dissolved in water (10 cm³) and a sample placedon a microscope. Almost monodispersed spherical entities of ˜3 μmdiameter were observed (FIG. 1).

EXAMPLE 2—PREPARATION OF SELF-ASSEMBLED MICROPARTICLES

Brassylic acid (1.54 g, 6.31 mmol) and dimethylaminoethanol (DMAE, 1.12g, 12.62 mmol) were dissolved in water (10 cm³) and a sample placed on amicroscope. Almost monodispersed spherical entities of ˜3 μm diameterwere observed.

EXAMPLE 3—PREPARATION OF SELF-ASSEMBLED MICROPARTICLES

Brassylic acid (1.54 g, 6.31 mmol) and 4-methylmorpholine (NMM, 1.275 g,12.62 mmol) were dissolved in water (10 cm³) and a sample placed on amicroscope. Almost monodispersed spherical entities of ˜3 μm diameterwere observed.

EXAMPLE 4—PREPARATION OF SELF-ASSEMBLED MICROPARTICLES

The above dicarboxylic acid dissolution experiments were also carriedout using a range of acids and a range of water soluble organic bases.Some of the combinations tested are listed below. The combinations hadan acid group to basic group molar ratio of 0.9 to 1.1:1. All of thesecombinations formed the spherical entities as described in Example 1.

-   Pimelic acid plus NMM-   Suberic acid plus NMM-   Azelaic acid plus NMM-   Sebacic acid plus NMM-   Sebacic acid plus DMAP-   Sebacic acid plus DMAE-   Sebacic acid plus imidazole-   Dodecanedioic acid plus NMM-   Dodecanedioic acid plus DMAP-   Dodecanedioic acid plus DMAE-   C36 dimer acid plus NMM

EXAMPLE 5—POLY(DIALLYLDIMETHYLAMMONIUM CHLORIDE) (PDAC) SPHERISOMES

PDAC (1.615 g, 10 mmol) was dissolved in water (50 cm³) and NaOH (0.4 g,10 mmol) added. Brassylic acid (1.22 g, 5 mmol) was added to thissolution and allowed to dissolve overnight. This appeared by visualinspection to be a clear solution but was confirmed to be a suspensionof ˜3 μm microparticles when observed under the microscope. A microscopephotograph is shown in FIG. 2.

EXAMPLE 6—PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLESCONTAINING HAEMOGLOBIN

PDAC (16.167 g of 20% solution, 20 mmol) was dissolved in water (100cm³) and NaOH (0.8 g, 20 mmol) added. Brassylic acid (2.44 g, 10 mmol)was added to this solution and allowed to dissolve. Human haemoglobin(2.44 g) was dissolved in water (100 cm³) and added to the solution ofbrassylic acid/PDAC SpheriSomes. The mixture was filtered through a 0.5μm cartridge and a sample placed on a microscope. Microspheres of ˜3 μmdiameter were still present. A solution ofN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCl)(4.6g, 24 mmol) and 1-hydroxybenzotriazole (HOBt) (0.47 g, 1.2 mmol) weredissolved in water (50 cm³) and added to the above solution. Thecross-linking reaction was left overnight, the resultant particleswashed by tangential flow filtration (TFF). It was noted that thesupernatant was colourless confirming that the haemoglobin wasincorporated into the SpheriSomes. A sample of the SpheriSomes waslyophilised. FIG. 3 shows a scanning electron micrograph of theresultant microspheres.

EXAMPLE 7—PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLESCONTAINING HAEMOGLOBIN

PDAC (16.167 g of 20% solution, 20 mmol) was dissolved in water (100cm³) and NaOH (0.8 g, 20 mmol) added. Brassylic acid (2.44 g, 10 mmol)was added to this solution and allowed to dissolve. Human haemoglobin(3.66 g) was dissolved in water (100 cm³) and added to the solution ofbrassylic acid/PDAC SpheriSomes. The mixture was filtered through a 0.5μm cartridge and a sample placed on a microscope. Microspheres of ˜3 μmdiameter were still present. A solution of EDCl (4.6 g, 24 mmol) andHOBt (0.47 g, 1.2 mmol) were dissolved in water (50 cm³) and added tothe above solution. The cross-linking reaction was left overnight, theresultant particles washed by tangential flow filtration (TFF). It wasnoted that the supernatant was colourless confirming that thehaemoglobin was incorporated into the SpheriSomes. FIG. 4 shows amicroscope photograph of the resultant microspheres.

EXAMPLE 8—PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLESCONTAINING HAEMOGLOBIN

PDAC (16.167 g of 20% solution, 20 mmol) was dissolved in water (100cm³) and NaOH (0.8 g, 20 mmol) added. Brassylic acid (2.44 g, 10 mmol)was added to this solution and allowed to dissolve. Human haemoglobin(4.88 g) was dissolved in water (100 cm³) and added to the solution ofbrassylic acid/PDAC SpheriSomes. The mixture was filtered through a 0.5μm cartridge and a sample placed on a microscope. Microspheres of ˜3 μmdiameter were still present. A solution of EDCl (4.6 g, 24 mmol) andHOBt (0.47 g, 1.2 mmol) were dissolved in water (50 cm³) and added tothe above solution. The cross-linking reaction was left overnight, theresultant particles washed by tangential flow filtration (TFF). It wasnoted that the supernatant was slightly pink confirming the majority ofthe haemoglobin was incorporated into the SpheriSomes. FIG. 5 shows amicroscope photograph of the resultant microspheres.

EXAMPLE 9—PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLESCONTAINING HAEMOGLOBIN

L-Carnitine (1.612 g, 10 mmol) was suspended in water (20 cm³) and NaOH(0.4 g, 10 mmol) added. Brassylic acid (1.22 g, 5 mmol) was added tothis solution and the mixture allowed to dissolve. Human haemoglobin(1.83 g) was dissolved in water (200 cm³) and added to the solution ofBrassylic acid/carnitine SpheriSomes. The mixture was filtered through a0.5 μm cartridge and a sample placed on a microscope. Microspheres of ˜3μm diameter were present. A solution of EDCl (4.6 g, 24 mmol) and HOBt(0.37 g, 2.4 mmol) were dissolved in water (25 cm³) and added to theabove solution. The cross-linking reaction was left overnight, theresultant particles washed by tangential flow filtration (TFF). It wasnoted that the supernatant was colourless confirming that thehaemoglobin was incorporated into the SpheriSomes.

EXAMPLE 10—PREPARATION OF CROSS-LINKED SELF-ASSEMBLED MICROPARTICLESCONTAINING HAEMOGLOBIN

Tetraethyl ammonium hydroxide (TEA.OH) (4.2 cm³ of a 35% solution, 10mmol) was added to water (20 cm³) and Brassylic acid (1.22 g, 5 mmol)was added to this solution. The Brassylic acid dissolved immediately toform SpheriSomes. Human haemoglobin (1.83 g) was dissolved in water (200cm³) and added to the solution of Brassylic acid/TEA.OH SpheriSomes. Themixture was filtered through a 0.5 μm cartridge and a sample placed on amicroscope. Microspheres of ˜3 μm diameter were still present. Asolution of EDCl (2.3 g, 12 mmol) and HOBt (0.18 g, 1.2 mmol) weredissolved in water (25 cm³) and added to the above solution. Thecross-linking reaction was left overnight, the resultant particleswashed by tangential flow filtration (TFF). It was noted that thesupernatant was colourless confirming that the haemoglobin wasincorporated into the SpheriSomes.

1. A blood substitute product comprising mammalian haemoglobin and a self-assembled microparticle in which the microparticle comprises an acid having two or more acid groups and an organic base which is soluble in a hydrophilic solvent.
 2. A blood substitute product according to claim 1 in which the blood substitute product comprises a stable polymerized haemoglobin solution, comprising mammalian haemoglobin cross-linked with a self-assembled microparticle.
 3. A blood substitute product according to claim 1 in which the microparticle has a particle size of 0.5 to 10 microns.
 4. A blood substitute product according to claim 1 in which the molar ratio of acid groups to basic groups in the acid and base is from 0.6 to 1.4:1.
 5. A blood substitute product according to claim 1 in which the molar ratio of acid groups to basic groups is from 0.7 to 1.3:1.
 6. A blood substitute product according to claim 1 in which the microparticle comprises an acid having two or more acid groups and an organic base obtainable by a process comprising contacting the bis-acid and organic base in a hydrophilic solvent, wherein the acid is insoluble or sparingly soluble in the hydrophilic solvent and the organic base is soluble in a hydrophilic solvent.
 7. A blood substitute product according to claim 6 in which the solvent comprises an aqueous solution.
 8. A blood substitute product according to claim 6 wherein the solvent comprises a water in oil emulsion within an aqueous phase.
 9. A blood substitute product according to claim 1 in which the acid comprises a bis-acid.
 10. A blood substitute product according to claim 9 in which the acid comprises a bis-aliphatic acid.
 11. A blood substitute product according to claim 1 in which the acid comprises a bis-carboxylic fatty acid in which terminal carboxylic acids are linked by a region which is hydrophobic.
 12. A blood substitute product according to claim 1 in which the acid groups are separated by a saturated, or unsaturated aliphatic chain; or substituted saturated, or unsaturated aliphatic chain.
 13. A blood substitute product according to claim 1 in which the acid comprises a compound of general formula HOOC—(CH₂)_(n)—COON wherein n is sufficiently large that the bis acid is sparingly soluble or insoluble in water.
 14. A blood substitute product according to claim 13 in which n is at least 5 and not more than
 40. 15. A blood substitute product according to claim 1 in which the acid comprises brassylic acid, sebacic acid and/or azelaic acid.
 16. A blood substitute product according to claim 1 in which the organic base comprises an aliphatic amine or an aromatic amine having a basic character or other nitrogen-containing base.
 17. A blood substitute product according claim 1 in which the organic base comprises one or more of an alkylated amine and an alkylated polyamine.
 18. A blood substitute product according to claim 1 in which the organic base comprises one or more of N-methylmorpholine, N, N-dimethylaminoethanol, 4-dimethylaminopyridine, imidazole, 1-methylamidazole poly(diallyldimethylammonium chloride) (PDAC), didecyldimethylammonium chloride (DDAC), dodecyldipropylenetriamine (DDPT) and poly epsilon lysine.
 19. A blood substitute product according to claim 1 in which the microparticle comprises a multi-lamellar structure.
 20. A blood substitute product according to claim 1 in which the acid is reacted with haemoglobin to form a cross-linked species. 21.-23. (canceled) 